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Gene therapy using adenoviral vectors
Gene therapy using adenoviral vectors Bruce C Trapnell and Mario Gorziglia G e n e t i c T h e r a p y Inc, Gaithersburg and G e o r g e t o w n U n i v e r s i t y School of M e d i c i n e , W a s h i n g t o n DC, USA Growing interest in adenoviral gene-transfer vectors, stimulated by efforts to develop in vivo gene therapy for cystic fibrosis, has led to an evaluation of their use in many other applications of in vivo gene therapy. Studies are beginning to define strategies for the efficient, albeit transient, expression of the transferred gene and have further identified and partially characterized important host responses to in vivo gene transfer that modulate the duration of expression of the transgene. Ongoing work is actively exploring these issues, with a view to the design of the next generation of adenoviral vectors. Current Opinion in Biotechnology 1994, 5:61 7-625
Introduction Adenoviral vectors represent an important class of dehvery vehicle for in vivo gene therapy of diseases where the tissue to be treated cannot be removed, gene-corrected ex vivo and reimplanted. Cystic fibrosis (CF) provides such an example and has stimulated evaluation and development of the adenoviral vector system for clinical use. Several factors account for recent growing interest in adenoviral vectors. First, they can be easily rendered replication deficient by deletion of critical viral regulatory genes [1,2]. Second, they are efficient in vivo gene delivery vehicles. Third, they transduce both dividing and non-dividing cells. Finally, they can be produced easily and at high titers (1011-1012 infectious units per ml). Adenovirus-mediated gene transfer has now been evaluated in vai~]ous tissues and organs in animals and in several clinical trials in humans. One specialized role of adenoviral vectors for gene transfer involves their use as vaccines [3]. Adenoviruses have also been used in a new type of 'molecular conjugate' gene delivery vector (reviewed in [4°°]). This review focuses on current studies of the efficacy and safety of adenovirus vectors for in vivo gene transfer.
Structure and biology of human adenoviruses Human adenoviruses (reviewed in [1]) are nonenveloped DNA viruses. Nearly 50 serotypes of adenovirus have been identified, but only serotypes 5 (Ad5)
and 2 (Ad2) have been utilized extensively in strategies for gene therapy. These serotypes belong to a group of adenoviruses that causes an acute febrile upper respiratory illness accounting for 5-15% of occurrences of the 'common cold'. In support of their safe use for human gene therapy, adenoviruses have been evaluated extensively as live vaccines in millions of individuals and have never been associated with any type of tumor in humans. The adenovirus genome is 36 kb long, linear and includes four distinct early regions (El-E4) and an alternatively spliced major late region consisting of five components (L1-L5) [1]. E1 region proteins have several functions, including regulation of most other viral transcriptional units either directly or indirectly. The E2 region encodes proteins involved directly in adenoviral DNA replication. Specifically, E2a encodes a protein with important DNA-binding and regulatory functions [5], whereas E2b encodes a DNA polymerase and the terminal binding protein [1]. The E3 region is a cassette of genes encoding protein products that are involved in viral mechanisms to permit infected cells to evade the host immune system. The E4 region encodes proteins with multiple functions, including control of viral transcription, DNA replication and shut-off of host cell protein expression. The major late region encodes most of the viral structural proteins, including the capsid components, such as hexon and fiber. In wild-type adenovirus, E1 genes are actively transcribed immediately upon entry of the genome into the nucleus as a result of the activity of ubiquitous cellular transcription factors. E1 gene products then activate the
Abbreviations Ad2/S--adenovirus serotype 2/5; otlAT--ctl-antitrypsin; Avl--first-generation adenoviral vector; Av2--second-generation adenoviral vector; CI=~ystic fibrosis; CFTR--CF transmembrane conductance regulator; CMV---cytomegalovirus; CTL---cytotoxic T lymphocyte; IL--interleukin; LDL--Iow density lipoprotein; mdx--X-chromosome linked muscular dystrophic; NIH--National Institutes of Health; OTC--ornithine transcarbamylase; PAH--phenylalanine hydroxylase; pfu--plaque-forming units; RAC--Recombinant DNA Advisory Committee; RSV--Rous sarcoma virus.
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Mammaliangene studies remainder of the genetic program of the virus in a cascade. This cascade leads sequentially to DNA replication (beginning after about 8 hours), expression ofadenovirus late genes, production of infectious virus progeny and finally death of the cell and release of newly made virus. Recombinant replication-deficient first-generation adenoviral (Avl) vectors have an extensive deletion of El, which by blocking transcriptional upregulation and the cascade leading to virion replication and cell death, resuits in severe attenuation [2]. As this deletion renders Avl vectors replication-deficient, they must be produced in 293 cells, which express Ad5 E1 genes that trans-complement the E1 deficit. The E3 region is not necessary for replication and is also frequently [6,7",8], but not always [9], deleted. In most Avl vectors, the heterologous non-adenoviral minigene to be delivered (referred to as the transgene) is located within the E1 region deletion [2]. The combination of the E1 and E3 region deletions provides sufficient genomic space for insertion of approximately 7 kb of exogenous DNA [2]. Vectors with a large genome size approaching 105% of the normal size (-36kb), however, replicate poorly in vitro compared with wild-type virus [9], probably due to less efficient packaging of the large genome.
Efficacy of in vivo adenovirus-mediated gene transfer The efficacy of delivery and expression of the transgene to various organs in vivo is perhaps best considered separately with respect to the route of administration (see Table 1). Because of interest in the development of gene therapy for the fatal pulmonary component of CF, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lung has received significant attention in a number of laboratories. The ability of an Avl vector expressing normal human C F T R cDNA to correct the CF epithelial cell chloride ion secretory defect was first demonstrated by Rosenfeld et al. [6] and later confirmed by others [7"',8,9]. Many pre-clinical studies have also used 'reporter' vectors to assess transgene delivery and expression in a variety of animal models. These include the cotton rat, mouse, hamster, rabbit, sheep, ferret, nonhuman primates, and a xenograft of human respiratory epithelium reconstituted in a denuded rat trachea and implanted in a nude mouse (see Table 1). One of the most frequently utilized Avl reporter vectors expresses a nuclear-localizing ~-galactosidase enzyme that, in the presence of X-gal, stains the nucleus of transduced cells blue [10"]. Using this reporter, Yei et al. [11"] have observed transgene expression in up to 80% of the respiratory epithelium in exposed animals, akhough expression was variable and patchy. Avl vectors have been shown to transduce all major respiratory airway epithelial cell types [10",11"] as well as cells within the submucosal glands [7"]. This is important because the exact site leading to
the fatal lung pathology in CF has not been determined. Luciferase is a second reporter that has been used effectively for quantitative studies of the efficacy of single and repeated administration [12"]. Despite the high efficiency of transgene delivery to the lung in many models, the duration of [~-galactosidase expression has been found to be transient. Some reports describe expression duration times in the range of approximately two to three weeks, whereas others describe substantially longer expression (see Table 1). Rosenfeld et al. [6] demonstrated Avl vector derived m R N A expression of a C F T R transgene in the lung at six weeks using reverse transcription, polymerase chain reaction (PCR) amplification and Southern hybridization. In another report, Engelhardt et al. [7"*], using the human bronchial xenograft model in nude mice, have demonstrated that an Avl vector transduces 5-20% of human surface airway epithelial cells. Importantly, transgene expression in these immunocompromized animals lasts longer (for about three months), suggesting that the host response (see below) may reduce the duration of transgene expression [13"']. Avl-mediated in vivo transduction of the liver is the next best studied model. An interesting and useful observation, albeit poorly understood, is that hepatocytes can be efficiently transduced by intravenous tail-vein injection of Avl vector in mice. Injection of a vector expressing human factor IX results in hepatocyte transduction levels sufficient to generate therapeutic levels of biologically active factor IX [14]. Evaluation of the kinetics of expression has demonstrated a peak level of expression within one week of injection followed by a steady decrease, but measurable plasma levels for up to nine weeks. A similar vector has been used successfully to correct the bleeding diathesis ofhemophilic dogs, although this correction is only transient [15"]. In another model of liver-targeted disease, Ishibash et al. [16"] have tested the efficacy of an Avl vector expressing the human low density lipoprotein (LDL) receptor to correct the hypercholesterolemia of mice carrying a double knockout of the LDL receptor gene. Intravenous injection of the vector lowered plasma cholesterol levelsPsigflificantly, albeit transiently. Furthermore, the same vector also substantially lowered cholesterol levels in normal mice. Two strains of mice (Sfp-Ash and C 3 H / H E N C R M T V ) that lack functional ornithine transcarbamylase (OTC) in the liver have been gene-corrected in vivo by Avl vectors expressing the O T C gene [17",18]. Fang et al. [19] have demonstrated correction of phenyketonuria in the phenylalanine hydroxylase (PAH) deficient P A H em~2 mouse using an Avl vector to transfer the PAH cDNA to the liver in vivo. Avl vectors have been evaluated in models for in vivo gene transfer to various other sites, including the brain, vascular endothelium, muscle, gall bladder, bone marrow, ocular, peritoneal, and synovial tissues (Table 1). In the central nervous system, studies have demonstrated
Gene therapy using adenoviral vectors Trapnell and Gorziglia Table 1. Studies reporting gene transfer and duration of transgene expression in various organs and tissues. Gene transfer target
Transgene*
Reference
Macaque
CFTR (3--42) lacZ (1-21 ) s l A T (7) CFTR (3-21)t lacZ (3-70)t CFTR (42) lacZ (6-60) CFTR (4) lacZ (4) CFTR (21)
[11 ",36] [10"] [28] [41 ,,] [41 " , 4 2 " ] [36"] [37,38] [45] [4S] (RW Wilmott et al., unpublished data)
Human
CFTR (9-21)
[43"',44"]
Human Rhesus monkey
CFTR (4) CFTR (18)
[44"] [36"]
Bronchial epithelium (xenograft)
Human
CFTR (35)
[7"']
Liver
Mouse
lacZ (7-70) OTC (4~150) LDL (4) Factor IX (70) TNF inhibitor (42) IL-6 (6) lacZ (21 ) CFTR (3) Factor IX (60)
[42",46] [I 7",I 8"] [I 6"] [14] [47] [48] [49] [49] [I 5 " ]
Mouse
lacZ (75-360) dystrophin (90-I 80)
[30] [31,32"]
Rat
CAT (55)
[33]
Rat
~1 AT (7)
Mouse
lacZ (45-60) lacZ (56)
[20] [21,22] [23]
Eye
Mouse
lacZ (50)
[50]
Synovium
Rabbit
lacZ (56)
[51 ]
Vascular wall
Sheep
lacZ (15) ~I AT (I 5) lacZ (I 4)
[24] [24] [25]
Location
Species
Lung
Cotton rat
Mouse Rhesus monkey Baboon
Nasal epithelium Bronchial epithelium
Rat
Dog Skeletal muscle
Heart Brain
"
Rat Hepatoc,/tes
Human
LDL (2) OTC (15)
[52] [17"]
Macrophages
Human
lacZ (I 4)
[53]
Intraperitoneal
Cotton rat
0~IAT (24)
[27"]
*Duration of transgene expression (days) is given in parentheses, tAlso includes data for Av2 vectors.
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620
Mammaliangene studies reporter transgene expression in myelinated neurons, oligodendrocytes, astrocytes and ventricular ependymal cells [20-23]. In one study, detectable transgene expression was seen in the brain for up to two months [22]. In vivo gene transfer to the vascular wall endothehum is an important potential application for adenoviral vectors. Another therapeutic target is the restenosis that frequently follows percutaneous transluminal coronary angioplasty. In this context, several investigators have demonstrated Avl vector-mediated transgene expression in endothelium, media and adventitia of the vascular wall [24,25,26°]. Many of these studies have used double vessel ligation to deliver vector locally and thereby achieve efficient gene transfer to the vascular wall [25,26°]. This is not clinically feasible, however, and other localization methods with a variety of catheter types are under evaluation. One interesting approach for increasing the efficiency of Avl vector-mediated gene delivery to vascular cells involves increasing the apparent rate ofgene transfer using a biocompatible compound known as poloxamer 407 [27°°]. Delivery of genes for replacement of plasma proteins such as 0t-antitrypsin, initially accomplished by delivery to pulmonary epithelium [28], has now been demonstrated after gene transfer to vascular endothelium [24] or abdominal peritoneum [29]. Avl vectors have been used successfully for in vivo gene transfer to both skeletal and cardiac muscle [30,31,32°°,33]. Using intravascular injection, one group reported ~-galactosidase expression that persisted for 12 months in both cardiac and skeletal muscle [30]. Other studies have used an intramuscular approach. For example, Ikagot et al. [31] employed an Avl vector expressing a human minidystrophin gene injected into the bicep femoris of the X-chromosome linked muscular dystrophic (mdx) mouse. In this model, transgene expression persists for at least three months. In another study, Kass-Eisler et al. [33] have administered an Avl vector expressing a chloramphenicol reporter to rats by cardiac ventricular wall injection. They found that, al-
though transduction of cardiac myocytes was patchy, in some regions, nearly 100% of the myocytes were transduced. Gene expression peaked at five days, but was still detectable 55 days after administration. Therapeutic approaches for acquired disorders, such as cancer, have also been explored using two different strategies: 'tumor vaccination', which enhances existing host anti-tumor immunity; and 'tumor sensitization', which creates tumor cell specific cytotoxicity. Using the first approach, an Avl vector containing an interleukin (IL)-2 transgene has been shown to enhance anti-tumor immunity against P815 cells, a tumor cell fine, implanted subcutaneously in nude mice [34]. Using the second appraoch, Chen et al. [35] have utilized a combination of a herpes thymidine kinase gene and ganciclovir to successfully treat pre-established tumors in a murine intrathecal brain tumor model.
Host response to in vivo adenovirus-mediated gene transfer Evaluation of the safety of Avl vectors has been carried out predominantly as part of pre-clinical safety studies for vectors developed for the clinical treatment of CF lung disease. Although safety questions were not considered in initial assessments of the efficacy of in vivo Avl vector-mediated gene delivery [6,28], several studies have now demonstrated and characterized, in part, the host response to in vivo Avl administration [8,11°,12°,36°°,37,38,39"°,40]. These studies (summarized and referenced in Table 2) have investigated the effects of Avl vector delivery to the lung or liver on non-specific inflammation within the target organ, the humoral response, the anti-vector antibody response and vector-induced cellular immunity. Using the cotton rat model first used for efficacy studies, Yei, et al. [11 °] have demonstrated that acute cellular
Table 2. Host-vector interactions observed with adenoviral vector mediated in vivo gene transfer. Adenovirus vector Generation
Avl Avl Avl Avl Avl Avl Avl Avl Avl Av2 Av2
Vector administration
7
z
Host response
Reference
Genotype
Promoter/ transgene*
Dose (pfu)
Route
Species
Organ
Inflammation
Immune response
Duration (days)
Ad5 EI-/E3AdS El-/E3AdS El-/E3AdS El-/E3Ad5 El-/E3Ad5 El-/E3Ad5 EI-/E3Ad5 E1-/E3÷ Ad2 EI-/E3÷ Ad5 EI-/E3-/E2a ts Ad5 EI-/E3-/E2a ts
EIa/RSV-IacZ CMV/~actin-lacZ CMV/~actin-lacZ CMV/~actin-lacZ
109 10I0 1010 109 2 x 109 10 I] 1010 2.5x 109 4.1 x l O 9 2x109 109
Inhalation Billiary tract Billiary tract Intravenous Tracheal Bronchial Instillation Instillation Tracheal Tracheal Intravenous
Cotton rat Mouse (CBA) Mouse (nu/nu) Mouse (CBA) Mouse (CBA) Rhesus Baboon Rhesus Cotton rat Mouse (CBA) Mouse (CBA)
Lung Liver Liver Liver Lung Lung Lung Nose Lung Lung Liver
Yes Yes ND** Yes Yes Yes Yes No No Yes Yes
Yes, humoral Yes, cellular ND** Yes, cellular Yes, cellular Yes, humoral ND** Yes, humoral Yes, humoral Yes, cellular Yes, cellular
3 21
[11°] [13ee]
60 7 7
[13ee] [42 ee] [41 oe]
60
[3B]
CMV/13actin-lacZ
EIa/MLP-CFTR CMV/~actin-CFTR Ela~Ia-CFTR Ela/Ela-CFTR CMV/~actin-lacZ CMV/l~actin-lacZ
*Each entry indicates the combination of expression cassette elements, including enhancer/promoter-transgene. **ND indicates analysis not done
21 18
[30]
42
[36°°] [36°e]
21 70
[41 eel [42 °°]
Gene therapy using adenoviral vectors Trapnell and Gorziglia inflammation is seen within the pulmonary parenchyma one day after administration o f a C F T R - e x p r e s s i n g Avl vector (AvlCf2) to the lungs. The inflammatory response peakes at day three to day four and decreases thereafter over the course of three to four weeks. Initially, the inflammatory response consisted ofneutrophil-dominated, perivascular, peribronchiolar and intra-alveolar infiltrates, with occasional neutrophil infiltration into airway epithelium. A lymphocytic infiltrate, predominantly in a peribronchiolar and perivascular distribution, develops after seven to ten days and is accompanied by increased numbers of alveolar macrophages. This host response is clearly transient, dose-dependent and significantly less pronounced than the response to wild-type replicative adenovirus. A similar pattern o f non-specific neutrophilic inflammation followed by lymphocytic immune response has been observed with vectors expressing different transgenes, suggesting that the vector itself is most important [11°]. In another study, Zabner et al. [36 °°] have reported a less dramatic host response in cotton rats following administration o f an Avl vector containing an intact E3 region. The reason for this apparent difference in host response between E3- and E3 + Avl vectors is unclear because transactivation o f the E3 region promoter is dependent on E l , which is absent from these types of vector. Studies in non-human primates, including the baboon [39°'], rhesus monkey [38] and macaque (RW Wilmott et al., unpublished data), have demonstrated host responses similar in character to those observed in cotton rats, but with perhaps a more protracted timecourse. Adult baboons receiving 109-1010 pfu per animal o f an
E 1 - / E 3 - Avl vector by bronchoscopic instillation remain clinically well, but show roentgenographic infiltration as well as significant parenchymal cellularity, which peaks 3 weeks after administration [39°°]. Macaques receiving 1010pfu per animal o f an E 1 - / E 3 - A v l vector, delivered bronchoscopically to a single lung lobe, similarly showed no adverse clinical findings, but did present a similar histologic pattern in the lung (R.W Wilmott et al., unpublished data). In rhesus monkeys, intranasal administration o f 2 . 8 x 109pfu per animal of an E1-/E3 + Avl vector resulted in no local or systemic inflammatory response, but did result in anti-vector antibodies [36°°]. Another study in rhesus monkeys reported no histopathology after bronchoscopic delivery of 5 x l 0 9 p f u per animal o f an E 1 - / E 3 - Avl vector to the main bronchi [37]. A third study, in which rhesus monkeys were given 1011 pfu per animal of an Avl vector to the main bronchi, demonstrated histopathology similar to that seen in cotton rats and baboons [38]. A humoral antibody response to Avl vector has been demonstrated in a variety o f animal models, including the cotton rat [12°*], mouse [13°°], baboon [48], macaque [39 °°] and rhesus monkey [36°,37,38]. Pulmonary administration of Avl vectors to cotton rats [12 °°] or macaques (RM Wilmott et al., unpublished data) induces a neutralizing antibody response that is dose-dependent, suggesting an absence of in vivo vector replication. As a control, cotton rats receiving wildtype replicative Ad5 show a similar high-titer antibody response, regardless o f the initial dose [12°°]. Zabner et al. [36°°] have reported that repeated administration o f Avl vectors in cotton rats and rhesus monkeys at rel-
Table 3. Approved Phase I clinical trials of adenovirus-mediated gene therapy for cystic fibrosis lung disease. Vector
Target organ
Dose range (pfu)
Number of patients*
Clinical study site
Principal investigator(s) and references
Ad-CFTR
Nose Lung
2 x 105-2 x 108 2 x 107-2 x 1010
14 10
NIH/University of Cornel l
Crystal [54]
Ad2/CFTR-1
Nose
2 x 106-5 x 107
3
University of Iowa
Welsh [55]
Ad.CB-CFTR
Lung
2.1 x 107-2.1 x1010
20
University of Michigan
Wilson [56]
Avl Cf2
Nose Lung
105-108 106-108
15 15
University of Cincinnati
Wilmott, Whitsett and Trapnell [57,58]
Ad.CB-CFTR
Nose
2 x 108-101!
12
University of North Carolina at Chapel Hill
Boucher and Knowles [59]
Maxillary sinus
2x 107-2x 1010
10-20
University of Iowa
Welsh and Smith**
Ad2-ORF6/PGK-/CFTR-2
*Patient numbers in individual trials are as updated in the Data Management Report from the RAC. **Approved by the National Institutes of Health, February 10th, 1994, Protocol #9312-067
621
622
Mammaliangene studies atively low doses results in successful gene transfer and expression for up to six weeks. Even so, another study has demonstrated that the efficiency of transduction and gene expression after repeat administration is inversely correlated with the humoral antibody response to the vector [12"]. Intravascular administration of Avl vectors results in reporter gene expression in the liver for 60 days in immune deficient athymic (nu/nu) mice compared with only 21 days in normal (CBA) mice, suggesting that a T-lymphocyte response is an important determinant o f the duration o f transgene expression [13**,41°°]. Immunological detection o f low levels of adenoviral hexon and fiber protein supports the concept that basal expression of such adenoviral late gene products in the liver of these animals might provide a basis for a specific cellular immune response directed at the transduced cells [13°°]. This and subsequent studies have verified the existence o f a cytotoxic lymphocyte (CTL) response to adenovirus proteins in animals receiving Avl vectors [13°°]. These observations are supported by studies in normal cotton rats immunocompromized artificially by administration o f dexamethasone or cyclosporin A (S Yei, B Trapnell, unpublished data). O n the basis o f the above observations, a second-generation adenoviral (Av2) vector has now been constructed. A potential problem with Avl vectors is that low-level expression o f adenoviral late genes probably induces an immune attack directly on the transduced cells. As expression of adenoviral late genes is strongly associated with adenoviral D N A replication, the Av2 vector has been designed not only to have deletions in E1 and E3 (similar to Avl), but also to have an additional singlebase substitution in the gene encoding the DNA-binding protein (DBP). This mutation is identical to Ad5ts125, a temperature-sensitive adenovirus mutant with a defect in DBP that renders the virus unable to replicate at 39°C [1,5]. Thus, at a low (permissive) temperature (32°C), the DBP is able to function normally, whereas at the non-permissive temperature (39°C; the core temperature o f rodents), the DBP is non-functional. The inclusion of this mutation in the Av2 vector reduces adenoviral D N A synthesis, late gene expression and cripples replication at the non-permissive temperature. In vivo evaluation o f this Av2 vector delivered to the liver o f normal CBA mice indicates a decreased C T L response [13"',42 °°] and an increased duration o f transgene expression.
Clinical trials with adenoviral vectors At the time of writing, six clinical trials o f human gene therapy using adenoviral vectors have been approved in the United States by the National Institutes o f Health (NIH) Recombinant DNA Activities Committee (RAC) and the Food and Drug Administration (FDA) (Table 3), and one trial has received approval in Europe. All o f these studies involve adenovirus-mediated gene transfer to the respiratory tract of individuals with CE
Final results of one trial involving nasal administration have demonstrated physiological correction o f the chloride secretory defect for three weeks [43•']. No adverse clinical findings linked to the vector itself have been demonstrated. Interim results of another trial have demonstrated the presence o f vector D N A in the nose (seven days after injection) and lung (15 days after injection), expression oftransgene m R N A in the nose at nine days, and C F T R expression in the nose (two days after injection) following vector administration [44°°]. In this study, no clinical, radiological or clinical laboratory findings were observed in patients receiving up to 2 × 107 pfu per patient to the lung. However, one patient receiving 2 × 109 pfu ofadenoviral C F T R in the lung developed an acute self-limited febrile response [44°°]. Although this was associated with a rise in anti-adenovirus antibody titer and lung IL-6 levels, the etiology o f this host-response could not be established definitively.
Conclusions The past year has seen significant and growing interest in the evaluation o f adenoviral vectors in a variety of models o f in uiuo gene therapy. As a result, much is now known concerning the molecular biology of these firstgeneration El-deleted vectors and the host responses that follow in vivo administration, both in a variety o f animal models and in humans. Despite efficient in uivo transgene delivery, expression is transient and appears to be limited by an inflammatory/immune response to the vector. The basis o f this response may include ongoing lowlevel constitutive expression o f adenoviral genes from the vector backbone leading to CTL-induced removal o f transduced cells. A second-generation vector capable o f prolonged transgene expression has been constructed, and it is hoped that this, and other improvements, will further reduce the host response, thus increasing the potential utility of adenoviral vectors.
Acknowledgements We would like to thank jennifer Lee and Soonpin Yei for help with the literature review, Paul Tolstoshev and Elizabeth Ashforth for c~itic}l review of the manuscript, and Gerri Smith for typing the article.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
Ginsberg HS: The adenoviruses. New York: Plenum Press; 1984.
2.
Trapnell BC: Adenoviral vectors for gene transfer. Adv Drug Del Rev 1993, 12:185-199.
3.
Both GW, Lockett LJ, Janardhana V, Edwards SJ, Bellamy AR, Graham FL, Prevec L, Andrew ME: Proteclive immunity to rotavirus-induced diarrhoea is passively transferred to newborn mice from naive dams vaccinated with a single dose of a recombinant adenovirus expressing rotavirus VP7sc. Virology 1993, 193:940-950.
G e n e therapy using adenoviral vectors Trapnell and G o r z i g l i a 4. Cotten M, Wagner E: Non-viral approaches to gene therapy. -• Curr Opin Biotechnol 1993, 4:705-710. A thorough review of the use of adenovirus and adenoviral vectors in molecular conjugate vectors. Such vectors take advantage of the adenoviral 'endosomolysis' mechanism to achieve efficient delivery of large transgene plasmids. 5.
Brough DE, Drouguett G, Horwitz MS, Klessig DF: Multiple functions of the adenovirus DNA-binding protein are required for efficient viral DNA synthesis. Virology 1993, 196:269-281.
6.
Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthai ER, Dalemans W, Fukayama M, Bargon J, Stier LE, StratfordPerricaudet L et aL: In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 1992, 68:143-155.
7. •*
Engelhardt )F, Yang Y, Stratford-Perricaudet LD, Allen ED, Kozarsky K, Perricaudet M, Yankaskas JR, Wilson JM: Direcl gene transfer of human CFTR into human bronchial epithelia of xenografts with El-deleted adenoviruses. Nature Genet 1993, 4:27-34. An important report of the creation of the human bronchial epithelial xenograft model for evaluation of gene transfer to human epithelial cells in an in vivo setting. 8.
Mittereder N, Yei S, Bachurski C, Cuppoletti J, Whitsett JA, Tolstoshev P, Trapnell BC: Evaluation of the efficacy and safety of in vitro adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther 1994, 5:717-729.
9.
Rich DP, Couture LA, Cardoza LM, Guiggio VM, Armentano D, Espino PC, Hehir K, Welsh MJ, Smith AE, Gregory RJ: Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum Gene Ther 1993, 4:461-476.
10. •
Mastrangeli A, Danel C, Rosenfeld MA, Stratford-Perricaudet L, Perricaudet M, Pavirani A, Lecocq JP, Crystal RG: Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J Clin Invest 1993, 91:225-234. Provides a clear demonstration that all of the five major cell types within the pulmonary airway surface epithelium are efficiently transduced targets for human adenovirus vectors. 11. •
Yei S, Mittereder N, Wert S, Whitsett ]A, Wilmott RW, Trapnell BC: In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung. Hum Gene Ther 1994, 5:733-746. This study demonstrates and defines a dose-dependent transient inflammatory host response in the cotton rat to in vivo pulmonary administration of human adenovirus vectors. 12. •.
Yei S, Mittereder N, Tank K, O'Sullivan C, Trapnell BC: Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung. Gene Ther 1994, 1:192-200. This paper and [36 ••] are among several papers addressing the feasibility of repeated administration of recombinant Avl vectors. This paper demonstrates that although re-administration to the lung is possible, the efficiency of repeat transduction and gene expression is inversely proportional to the host anti-adenoviral vector antibody response resulting from previous exposure to the vector. In view of the transient nature of adenovirus-mediated transgene expression, the feasibility of clinical use of these vectors depends critically on repeated exposures. 13. ••
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM: Cellular immunity to viral antigens limits El-deleted adenoviruses for gene therapy. Proc Nat/ Acad Sci USA 1994, 91:4407-4411. An important paper describing the T cell mediated host response to in vivo adenovirus administration. This CTL response appears to be directed at transduced cells, leading to their elimination, thus shortening the duration of transgene expression. This study provides a basis for the enhancement of the next generation of adenoviral vectors. 14.
Smith TA, Mehaffey MG, Kayda DB, Saunders JM, Yei S, Trapnell BC, McClelland A, Kaleko M: Adenovlrus mediated expression of therapeutic plasma levels of human factor IX in mice. Nature Genet 1993, 5:397-402.
15. •*
Kay MA, Landen CN, Rotbenberg SR, Taylor LA, Leland F, Wiehle S, Fang B, Bellinger D, Finegold M, Thompson AR et al.: In vivo hepatic gene therapy: complete, albeit transient, correction of factor IX deficiency in hemophilia B dogs. Proc Nat/ Acad Sci USA 1994, 91:2353-2357. Administration of a human factor IX adenovirus corrects the bleeding diathesis in hemophilic dogs. This study demonstrates the feasibility of adenoviral vector mediated gene therapy of a non-rodent animal model for blood dyscrasias resulting from a genetic disorder of liver function. 16. •
Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J: Hypercholesterolemia in low density lipoproteln receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J C/in Invest 1993, 92:883-893. These authors describe the in vivo correction of LDL receptor deficient mice using an adenoviral vector. Results demonstrate the feasibility of using an adenoviral vector for gene therapy of liver disorders. 17. •
Morsy MA, Alford EL, Bett A, Graham FL, Caskey CT: Efficient adenoviral-mediated ornithine transcarbamylase expression in deficient mouse and human hepatocytes. J C/in Invest 1993, 92:1580-1586. This study describes transient in vivo adenoviral vector mediated correction of the genetic deficiency of ornithine transcarbamylase in the C3H/HENCRMTV mouse, thus demonstrating the feasibility of use of an adenoviral vector for gene therapy for liver disorders. 18.
Stratford-Perricaudet LD, Levrero M, Chasse JF, Perricaudet M, Briand P: Evaluation of the transfer and expression in mice of an enzyme-encoding gene using a human adenovirus vector. Hum Gene Ther ]990, 1:241-256.
19.
Fang B, Eisensmith RC, Li XHC, Finegold MJ, Shedlovsky A, Dove W, Woo SLC: Gene therapy for phenylketonuria: phenotypic correction in a genetically deficient mouse model by adenovirus-mediated hepatic gene transfer. Gene Ther 1994, 1:247-254.
20.
Bajocchi G, Feldman SH, Crystal RG, Mastrangeli A: Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors. Nature Genet 1993, 3:229-234.
21.
Akli S, Caillaud C, Vigne E, Stratford-Perricaudet LD, Poenaru L, Perricaudet M, Kahn A, Peschanski MR: Transfer of a foreign gene into the brain using adenovirus vectors. Nat Genet 1993, 3:224-228.
22.
Le Gal la Salle G, Robert JJ, Berrard S, Ridoux V, StratfordPerricaudet LD, Perricaudet M, Mallet J: An adenovirus vector for gene transfer into neurons and glia in the brain. Science 1993, 259:988-990.
23.
Davidson 8L, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ: A model system for in vivo gene transfer into the central nervous system using an adenoviral vector. Nature Genet 1993, 3:219-223.
24.
Lemarchand P, Jaffe HA, Danel C, Cid MC, Kleinman HK, Stratford-Perricaudet LD, Perricaudet M, Pavirani A, LeCocq JP, Crystal RG: Adenovirus-medlated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells. Proc Nat/ Acad 5ci USA 1992, 89:6482-6486.
25.
Lee SW, Trapnell BC, Rade JJ, Virmani R, Dicheck DA: In vlvo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries. Circ Res 1993, 73:797-807.
26. •
Rome J), Shayani V, Flugelman MY, Newman KD, Farb A, Virmani R, Dichek DA: Anatomic barriers influence the distribution of in vlvo gene transfer into the arterial wall: modeling with microscopic tracer particles and verification with a recombinant adenoviral vector. Arterioscler Thromb 1994, 14:148-161. A serious attempt to evaluate the practical aspects of in vivo gene delivery. Data suggest that tissue penetration is one of the major limitations for in vivo gene therapy of solid tissues. 27. -,
March KL, Madison JE, Trapnell BC: Pharmacoklnetics of adenoviral vector mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy. Hum Gene Ther 1994, in press. Use of a biocompatible block co-polymer (termed poloxamer 407) can substantially increase the apparent transduction rate constant of an ade-
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M a m m a l i a n gene studies noviral vector. This has important implications for in vivo gene therapy of clinical problems, such as restenosis following percutaneous transluminal angioplasty of coronary vessels, where time available for in vivo transduction is limited by clinical parameters. 28.
Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M e t al.: Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 1991, 252:431-434.
29.
Setoguchi Y, Jaffe HA, Chu CS, Crystal RG: Intraperitoneal in vivo gene therapy to deliver 1-antltrypsin to the systemic circulation. Am J Respir Cell Mol Biol 1994, 10:369-377.
30.
Stratford-Perricaudet LD, Makeh I, Perricaudet M, Briand P: Widespread long-term gene transfer to mouse skeletal muscles and heart. J C/in Invest 1992, 90:626-630.
31.
RagotT, Vincent N, Chafey P, Vigne E, Gilgenkrantz H, Couton D, Cartaud J, Briand P, Kaplan JC, Perricaudet M, Kahn A: Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice. Nature 1993, 361:647-650.
32. *°
Vincent N, Ragot T, Gilgenkrantz H, Couton D, Chafey P, Gregoire A, Briand P, Kaplan JC, Kahn A, Perricaudet M: Long-term correction of mouse dystrophic degeneration by adenovirus-medlated transfer of a mlnldystrophin gene. Nature Genet 1993, 5:130-134. Administration of a minidystrophin gene to the mdx mouse corrects dystrophic muscle degeneration. This demonstrates the feasibility of adenoviral vector mediated gene delivery to correct genetic disorders of muscle.
41. *-
Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson }M: Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nature Genet 1994, 7:362-369. See [42"*].
42. *-
Engelhardt JF, Ye X, Doranz B, Wilson JM: Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci USA 1994, 91:6196-6200. One of several papers (see also [41**]) from the same laboratory describing a single base change within the adenoviral vector backbone that appears to prolong the duration of transgene persistence. This mutation lies within the adenoviral DNA-binding protein gene and confers the temperature-sensitive function to this protein. Presumably, this mutation also blocks the sequential cascade of genetic events leading to low-level adenoviral late gene expression in transduced cells thought to induce a specific cellular immune response. 43. **
Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ: Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993, 75:207-216. First published results reporting the clinical use of human adenovirus vectors to transfer a normal CFTR cDNA to nasal epithelium of individuals with CF. This study demonstrates that gene therapy resulted in a small physiological correction of the CF epithelial cell chloride ion system secretory in treated nasal epithelium. 44. °-
Crystal RG, McElvaney NG, Rosenfeld MA, Chu C, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C: Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nature Genet 1994, 8:42-51. Reports the results of the first administration of an adenoviral vector to the nose and lungs of individuals with CF. This study demonstrates biological expression of the transgene in nasal and bronchial epithelium. A patient who developed a mild acute, self-limited febrile episode following vector administration is also described.
33.
Kass-EislerA, Falck-Pedersen E, Alvira M, Rivera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA: Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA 1993, 90:11498-11502.
34.
Haddada H, Ragot T, Cordier L, Perricaudet M: Adenoviral IL-2 gene transfer into P815 tumor cells abrogates tumorigenicity and induces antitumoral immunity in mice. Hum Gene Ther 1993, 6:703-711.
45.
Engelhardt JF, Simon RH, Yang Y, Zepeda M, Pendleton SW, Doranz B, Grossman M, Wilson JM: Adenovirus-medlated transfer of the CFTR gene to lung of non-human primates: biological efficacy study. Hum Gene Ther 1993, 4:771-780.
35.
Chen S, Shine HD, Goodman JC, Grossman RG, Woo SLC: Gene therapy for brain tumors: regression of experimental gliomas by adenovlrus-medlated gene transfer in vivo. Proc Natl Acad $ci USA 1994, 91:3054-3057.
46.
Li Q, Kay MA, Finegold M, Stratford-Perricaudet LD, Wood SLC: Assessmentof recombinant adenoviral vectors for hepatic gene therapy. Hum Gene Ther 1993, 4:403-409.
47.
Kolls J, Peppel K, Silva M, Beutler B: Prolonged and effective blockade of tumor necrosis factor activity through adenovirus-medlaled gene transfer. Proc Natl Acad Sci USA 1994, 91:215-219.
48.
Bout A, Perricaudet M, Baskin G, Imler JL, Scholte BJ, Pavirani A, Valerio D: Lung gene therapy: in vivo adenovlrus-mediated gene transfer to rhesus monkey airway epithelium. Hum Gene Ther 1994, 5:3-10.
Braciak TA, Mittal SK, Graham FL, Richards CD, Gauldie J: Construction of recombinant human type 5 adenoviruses expressing rodent IL-6 genes. An approach to investigate in vivo cytokine function. J/mmunol 1993, 151:5145-5153.
49.
Brody SL, Metzger M, Danel C, Rosenfeld MA, Crystal RG: Acute responses of non-human primates to airway delivery of an adenovirus vector containing the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther 1994, 5:821-836.
Yang Y, Raper SE, Cohn JA, Engelhardt JF, Wilson JM: An approach ~or jtreating the hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc Nat/ Acad Sci USA 1993, 90:4601-4605.
50.
Mashhour B, Couton D, Perricaudet M, Briand P: In vivo adenovlrus-mediated gene transfer into ocular tissues. Gene Ther 1994, 1:122-126.
51.
RoesslerBJ, Allen ED, Wilson JM, Hartman JW, Davidson BL: Adenoviral-mediated gene transfer to rabbit synovium in vivo. J C/in Invest 1993, 92:1085-1092.
52.
Kozarsky K, Grossman M, Wilson JM: Adenovirus-mediated correction of the genetic defect in hepatocytes from patlenls with familial hypercholesterolemla. Somat Cell Mol Genet 1993, 19:449-458.
53.
Haddada H, Lopez M, Martinache C, Ragot T, Abina MA, Perricaudet M: Efficient adenovlrus-medlated gene transfer into human blood monocyte-derlved macrophages. Biochem Biophys Res Commun 1993, 195:1174-1183.
54.
Crystal RC (Principal investigator): Protocol of gene therapy of the respiratory manifestations of cystic fibrosis using a prelica-
36. **
Zabner J, Petersen DM, Puga AP, Graham SM, Couture LA, Keyes LD, Lukason MJ, St George JA, Gregory RJ, Smith AE, Welsh MJ: Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nature Genet 1994, 6:75-83. See [12*°]. 37.
38.
39. °*
Simon RH, Engelhardt JF, Yang Y, Epeda M, Pendleton SW, Grossman M, Wilson JM: Adenovlrus-medlated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum Gene Ther 1993, 4:771-780. An important safety study of human adenovirus vector administration to the lung in non-human primates. This paper helped to alert the scientific community to the importance of the host response to administration of human adenovirus to the lung. 40.
Rosenfeld MA, Chu C, Seth P, Danel C, Banks 1, Yoneyama K, Yoshimura K, Crystal RG: Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replicationdeficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther 1994, 5:331-342.
Gene therapy using adenoviral vectors Trapnell and Gorziglia tion deficient recombinant adenovirus to transfer the normal cystic fibrosis transmembrane conductance regular cDNA to the airway epithelium. Fed Reg 1992, 57:49584. 55.
Welsh MJ (Principal Investigator): Cystic fibrosis gene therapy using an adenovlrus vector: in vlvo safety and efficacy in the nasal epithelium. Fed Reg 1992, 57:49584.
56.
Wilson JM (Principal Investigator): Gene therapy of cystic fibrosis lung diseases using E1 deleted adenovlrus: a phase trial. Fed Reg 1992, 57:49584.
57.
Wilmott RW, whitsett IA, Trapnell BC (Principal Investigators): A Phase 1 study of gene therapy of cystic fibrosis utilizing a replication deficient recombinant adenovlrus vector to deliver the human cystic fibrosis transmembrane conductance cDNA to the airways. Fed Reg 1993, 58:8500-8501.
58.
WilmottRW, Whitsett JA, Trapnell BC: Gene therapy for cystic fibrosis utilizing a replication-deficient recombinant aden-
ovirus vector to deliver the human cystic fibrosis transmembrane conductance regulator cDNA to the airways. Hum Gene Ther 1994, 5:1019-1057. 59.
Boucher RC, Knowles MR (Principal Investigators):Gene therapy for cystic fibrosis using E1 deleted adenovirus: a Phase 1 trial in the nasal cavity. Fed Reg 1993, 58:850].
BC Trapnell and M Gorziglia, Genetic Therapy Inc, 938 Clopper Road, Gaithersburg, Maryland 20878, USA, BC Trapnell, Georgetown University Medical Center, Department of Pharmacology, 3900 keservoir Road, North West Wxshington, District of Columbia 20007-2195, USA. Author for correspondence: BC Trapnell at Genetic Therapy Inc.
625
Introduction Adenoviral vectors represent an important class of dehvery vehicle for in vivo gene therapy of diseases where the tissue to be treated cannot be removed, gene-corrected ex vivo and reimplanted. Cystic fibrosis (CF) provides such an example and has stimulated evaluation and development of the adenoviral vector system for clinical use. Several factors account for recent growing interest in adenoviral vectors. First, they can be easily rendered replication deficient by deletion of critical viral regulatory genes [1,2]. Second, they are efficient in vivo gene delivery vehicles. Third, they transduce both dividing and non-dividing cells. Finally, they can be produced easily and at high titers (1011-1012 infectious units per ml). Adenovirus-mediated gene transfer has now been evaluated in vai~]ous tissues and organs in animals and in several clinical trials in humans. One specialized role of adenoviral vectors for gene transfer involves their use as vaccines [3]. Adenoviruses have also been used in a new type of 'molecular conjugate' gene delivery vector (reviewed in [4°°]). This review focuses on current studies of the efficacy and safety of adenovirus vectors for in vivo gene transfer.
Structure and biology of human adenoviruses Human adenoviruses (reviewed in [1]) are nonenveloped DNA viruses. Nearly 50 serotypes of adenovirus have been identified, but only serotypes 5 (Ad5)
and 2 (Ad2) have been utilized extensively in strategies for gene therapy. These serotypes belong to a group of adenoviruses that causes an acute febrile upper respiratory illness accounting for 5-15% of occurrences of the 'common cold'. In support of their safe use for human gene therapy, adenoviruses have been evaluated extensively as live vaccines in millions of individuals and have never been associated with any type of tumor in humans. The adenovirus genome is 36 kb long, linear and includes four distinct early regions (El-E4) and an alternatively spliced major late region consisting of five components (L1-L5) [1]. E1 region proteins have several functions, including regulation of most other viral transcriptional units either directly or indirectly. The E2 region encodes proteins involved directly in adenoviral DNA replication. Specifically, E2a encodes a protein with important DNA-binding and regulatory functions [5], whereas E2b encodes a DNA polymerase and the terminal binding protein [1]. The E3 region is a cassette of genes encoding protein products that are involved in viral mechanisms to permit infected cells to evade the host immune system. The E4 region encodes proteins with multiple functions, including control of viral transcription, DNA replication and shut-off of host cell protein expression. The major late region encodes most of the viral structural proteins, including the capsid components, such as hexon and fiber. In wild-type adenovirus, E1 genes are actively transcribed immediately upon entry of the genome into the nucleus as a result of the activity of ubiquitous cellular transcription factors. E1 gene products then activate the
Abbreviations Ad2/S--adenovirus serotype 2/5; otlAT--ctl-antitrypsin; Avl--first-generation adenoviral vector; Av2--second-generation adenoviral vector; CI=~ystic fibrosis; CFTR--CF transmembrane conductance regulator; CMV---cytomegalovirus; CTL---cytotoxic T lymphocyte; IL--interleukin; LDL--Iow density lipoprotein; mdx--X-chromosome linked muscular dystrophic; NIH--National Institutes of Health; OTC--ornithine transcarbamylase; PAH--phenylalanine hydroxylase; pfu--plaque-forming units; RAC--Recombinant DNA Advisory Committee; RSV--Rous sarcoma virus.
© Current Biology Ltd ISSN 0958-1669
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Mammaliangene studies remainder of the genetic program of the virus in a cascade. This cascade leads sequentially to DNA replication (beginning after about 8 hours), expression ofadenovirus late genes, production of infectious virus progeny and finally death of the cell and release of newly made virus. Recombinant replication-deficient first-generation adenoviral (Avl) vectors have an extensive deletion of El, which by blocking transcriptional upregulation and the cascade leading to virion replication and cell death, resuits in severe attenuation [2]. As this deletion renders Avl vectors replication-deficient, they must be produced in 293 cells, which express Ad5 E1 genes that trans-complement the E1 deficit. The E3 region is not necessary for replication and is also frequently [6,7",8], but not always [9], deleted. In most Avl vectors, the heterologous non-adenoviral minigene to be delivered (referred to as the transgene) is located within the E1 region deletion [2]. The combination of the E1 and E3 region deletions provides sufficient genomic space for insertion of approximately 7 kb of exogenous DNA [2]. Vectors with a large genome size approaching 105% of the normal size (-36kb), however, replicate poorly in vitro compared with wild-type virus [9], probably due to less efficient packaging of the large genome.
Efficacy of in vivo adenovirus-mediated gene transfer The efficacy of delivery and expression of the transgene to various organs in vivo is perhaps best considered separately with respect to the route of administration (see Table 1). Because of interest in the development of gene therapy for the fatal pulmonary component of CF, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lung has received significant attention in a number of laboratories. The ability of an Avl vector expressing normal human C F T R cDNA to correct the CF epithelial cell chloride ion secretory defect was first demonstrated by Rosenfeld et al. [6] and later confirmed by others [7"',8,9]. Many pre-clinical studies have also used 'reporter' vectors to assess transgene delivery and expression in a variety of animal models. These include the cotton rat, mouse, hamster, rabbit, sheep, ferret, nonhuman primates, and a xenograft of human respiratory epithelium reconstituted in a denuded rat trachea and implanted in a nude mouse (see Table 1). One of the most frequently utilized Avl reporter vectors expresses a nuclear-localizing ~-galactosidase enzyme that, in the presence of X-gal, stains the nucleus of transduced cells blue [10"]. Using this reporter, Yei et al. [11"] have observed transgene expression in up to 80% of the respiratory epithelium in exposed animals, akhough expression was variable and patchy. Avl vectors have been shown to transduce all major respiratory airway epithelial cell types [10",11"] as well as cells within the submucosal glands [7"]. This is important because the exact site leading to
the fatal lung pathology in CF has not been determined. Luciferase is a second reporter that has been used effectively for quantitative studies of the efficacy of single and repeated administration [12"]. Despite the high efficiency of transgene delivery to the lung in many models, the duration of [~-galactosidase expression has been found to be transient. Some reports describe expression duration times in the range of approximately two to three weeks, whereas others describe substantially longer expression (see Table 1). Rosenfeld et al. [6] demonstrated Avl vector derived m R N A expression of a C F T R transgene in the lung at six weeks using reverse transcription, polymerase chain reaction (PCR) amplification and Southern hybridization. In another report, Engelhardt et al. [7"*], using the human bronchial xenograft model in nude mice, have demonstrated that an Avl vector transduces 5-20% of human surface airway epithelial cells. Importantly, transgene expression in these immunocompromized animals lasts longer (for about three months), suggesting that the host response (see below) may reduce the duration of transgene expression [13"']. Avl-mediated in vivo transduction of the liver is the next best studied model. An interesting and useful observation, albeit poorly understood, is that hepatocytes can be efficiently transduced by intravenous tail-vein injection of Avl vector in mice. Injection of a vector expressing human factor IX results in hepatocyte transduction levels sufficient to generate therapeutic levels of biologically active factor IX [14]. Evaluation of the kinetics of expression has demonstrated a peak level of expression within one week of injection followed by a steady decrease, but measurable plasma levels for up to nine weeks. A similar vector has been used successfully to correct the bleeding diathesis ofhemophilic dogs, although this correction is only transient [15"]. In another model of liver-targeted disease, Ishibash et al. [16"] have tested the efficacy of an Avl vector expressing the human low density lipoprotein (LDL) receptor to correct the hypercholesterolemia of mice carrying a double knockout of the LDL receptor gene. Intravenous injection of the vector lowered plasma cholesterol levelsPsigflificantly, albeit transiently. Furthermore, the same vector also substantially lowered cholesterol levels in normal mice. Two strains of mice (Sfp-Ash and C 3 H / H E N C R M T V ) that lack functional ornithine transcarbamylase (OTC) in the liver have been gene-corrected in vivo by Avl vectors expressing the O T C gene [17",18]. Fang et al. [19] have demonstrated correction of phenyketonuria in the phenylalanine hydroxylase (PAH) deficient P A H em~2 mouse using an Avl vector to transfer the PAH cDNA to the liver in vivo. Avl vectors have been evaluated in models for in vivo gene transfer to various other sites, including the brain, vascular endothelium, muscle, gall bladder, bone marrow, ocular, peritoneal, and synovial tissues (Table 1). In the central nervous system, studies have demonstrated
Gene therapy using adenoviral vectors Trapnell and Gorziglia Table 1. Studies reporting gene transfer and duration of transgene expression in various organs and tissues. Gene transfer target
Transgene*
Reference
Macaque
CFTR (3--42) lacZ (1-21 ) s l A T (7) CFTR (3-21)t lacZ (3-70)t CFTR (42) lacZ (6-60) CFTR (4) lacZ (4) CFTR (21)
[11 ",36] [10"] [28] [41 ,,] [41 " , 4 2 " ] [36"] [37,38] [45] [4S] (RW Wilmott et al., unpublished data)
Human
CFTR (9-21)
[43"',44"]
Human Rhesus monkey
CFTR (4) CFTR (18)
[44"] [36"]
Bronchial epithelium (xenograft)
Human
CFTR (35)
[7"']
Liver
Mouse
lacZ (7-70) OTC (4~150) LDL (4) Factor IX (70) TNF inhibitor (42) IL-6 (6) lacZ (21 ) CFTR (3) Factor IX (60)
[42",46] [I 7",I 8"] [I 6"] [14] [47] [48] [49] [49] [I 5 " ]
Mouse
lacZ (75-360) dystrophin (90-I 80)
[30] [31,32"]
Rat
CAT (55)
[33]
Rat
~1 AT (7)
Mouse
lacZ (45-60) lacZ (56)
[20] [21,22] [23]
Eye
Mouse
lacZ (50)
[50]
Synovium
Rabbit
lacZ (56)
[51 ]
Vascular wall
Sheep
lacZ (15) ~I AT (I 5) lacZ (I 4)
[24] [24] [25]
Location
Species
Lung
Cotton rat
Mouse Rhesus monkey Baboon
Nasal epithelium Bronchial epithelium
Rat
Dog Skeletal muscle
Heart Brain
"
Rat Hepatoc,/tes
Human
LDL (2) OTC (15)
[52] [17"]
Macrophages
Human
lacZ (I 4)
[53]
Intraperitoneal
Cotton rat
0~IAT (24)
[27"]
*Duration of transgene expression (days) is given in parentheses, tAlso includes data for Av2 vectors.
619
620
Mammaliangene studies reporter transgene expression in myelinated neurons, oligodendrocytes, astrocytes and ventricular ependymal cells [20-23]. In one study, detectable transgene expression was seen in the brain for up to two months [22]. In vivo gene transfer to the vascular wall endothehum is an important potential application for adenoviral vectors. Another therapeutic target is the restenosis that frequently follows percutaneous transluminal coronary angioplasty. In this context, several investigators have demonstrated Avl vector-mediated transgene expression in endothelium, media and adventitia of the vascular wall [24,25,26°]. Many of these studies have used double vessel ligation to deliver vector locally and thereby achieve efficient gene transfer to the vascular wall [25,26°]. This is not clinically feasible, however, and other localization methods with a variety of catheter types are under evaluation. One interesting approach for increasing the efficiency of Avl vector-mediated gene delivery to vascular cells involves increasing the apparent rate ofgene transfer using a biocompatible compound known as poloxamer 407 [27°°]. Delivery of genes for replacement of plasma proteins such as 0t-antitrypsin, initially accomplished by delivery to pulmonary epithelium [28], has now been demonstrated after gene transfer to vascular endothelium [24] or abdominal peritoneum [29]. Avl vectors have been used successfully for in vivo gene transfer to both skeletal and cardiac muscle [30,31,32°°,33]. Using intravascular injection, one group reported ~-galactosidase expression that persisted for 12 months in both cardiac and skeletal muscle [30]. Other studies have used an intramuscular approach. For example, Ikagot et al. [31] employed an Avl vector expressing a human minidystrophin gene injected into the bicep femoris of the X-chromosome linked muscular dystrophic (mdx) mouse. In this model, transgene expression persists for at least three months. In another study, Kass-Eisler et al. [33] have administered an Avl vector expressing a chloramphenicol reporter to rats by cardiac ventricular wall injection. They found that, al-
though transduction of cardiac myocytes was patchy, in some regions, nearly 100% of the myocytes were transduced. Gene expression peaked at five days, but was still detectable 55 days after administration. Therapeutic approaches for acquired disorders, such as cancer, have also been explored using two different strategies: 'tumor vaccination', which enhances existing host anti-tumor immunity; and 'tumor sensitization', which creates tumor cell specific cytotoxicity. Using the first approach, an Avl vector containing an interleukin (IL)-2 transgene has been shown to enhance anti-tumor immunity against P815 cells, a tumor cell fine, implanted subcutaneously in nude mice [34]. Using the second appraoch, Chen et al. [35] have utilized a combination of a herpes thymidine kinase gene and ganciclovir to successfully treat pre-established tumors in a murine intrathecal brain tumor model.
Host response to in vivo adenovirus-mediated gene transfer Evaluation of the safety of Avl vectors has been carried out predominantly as part of pre-clinical safety studies for vectors developed for the clinical treatment of CF lung disease. Although safety questions were not considered in initial assessments of the efficacy of in vivo Avl vector-mediated gene delivery [6,28], several studies have now demonstrated and characterized, in part, the host response to in vivo Avl administration [8,11°,12°,36°°,37,38,39"°,40]. These studies (summarized and referenced in Table 2) have investigated the effects of Avl vector delivery to the lung or liver on non-specific inflammation within the target organ, the humoral response, the anti-vector antibody response and vector-induced cellular immunity. Using the cotton rat model first used for efficacy studies, Yei, et al. [11 °] have demonstrated that acute cellular
Table 2. Host-vector interactions observed with adenoviral vector mediated in vivo gene transfer. Adenovirus vector Generation
Avl Avl Avl Avl Avl Avl Avl Avl Avl Av2 Av2
Vector administration
7
z
Host response
Reference
Genotype
Promoter/ transgene*
Dose (pfu)
Route
Species
Organ
Inflammation
Immune response
Duration (days)
Ad5 EI-/E3AdS El-/E3AdS El-/E3AdS El-/E3Ad5 El-/E3Ad5 El-/E3Ad5 EI-/E3Ad5 E1-/E3÷ Ad2 EI-/E3÷ Ad5 EI-/E3-/E2a ts Ad5 EI-/E3-/E2a ts
EIa/RSV-IacZ CMV/~actin-lacZ CMV/~actin-lacZ CMV/~actin-lacZ
109 10I0 1010 109 2 x 109 10 I] 1010 2.5x 109 4.1 x l O 9 2x109 109
Inhalation Billiary tract Billiary tract Intravenous Tracheal Bronchial Instillation Instillation Tracheal Tracheal Intravenous
Cotton rat Mouse (CBA) Mouse (nu/nu) Mouse (CBA) Mouse (CBA) Rhesus Baboon Rhesus Cotton rat Mouse (CBA) Mouse (CBA)
Lung Liver Liver Liver Lung Lung Lung Nose Lung Lung Liver
Yes Yes ND** Yes Yes Yes Yes No No Yes Yes
Yes, humoral Yes, cellular ND** Yes, cellular Yes, cellular Yes, humoral ND** Yes, humoral Yes, humoral Yes, cellular Yes, cellular
3 21
[11°] [13ee]
60 7 7
[13ee] [42 ee] [41 oe]
60
[3B]
CMV/13actin-lacZ
EIa/MLP-CFTR CMV/~actin-CFTR Ela~Ia-CFTR Ela/Ela-CFTR CMV/~actin-lacZ CMV/l~actin-lacZ
*Each entry indicates the combination of expression cassette elements, including enhancer/promoter-transgene. **ND indicates analysis not done
21 18
[30]
42
[36°°] [36°e]
21 70
[41 eel [42 °°]
Gene therapy using adenoviral vectors Trapnell and Gorziglia inflammation is seen within the pulmonary parenchyma one day after administration o f a C F T R - e x p r e s s i n g Avl vector (AvlCf2) to the lungs. The inflammatory response peakes at day three to day four and decreases thereafter over the course of three to four weeks. Initially, the inflammatory response consisted ofneutrophil-dominated, perivascular, peribronchiolar and intra-alveolar infiltrates, with occasional neutrophil infiltration into airway epithelium. A lymphocytic infiltrate, predominantly in a peribronchiolar and perivascular distribution, develops after seven to ten days and is accompanied by increased numbers of alveolar macrophages. This host response is clearly transient, dose-dependent and significantly less pronounced than the response to wild-type replicative adenovirus. A similar pattern o f non-specific neutrophilic inflammation followed by lymphocytic immune response has been observed with vectors expressing different transgenes, suggesting that the vector itself is most important [11°]. In another study, Zabner et al. [36 °°] have reported a less dramatic host response in cotton rats following administration o f an Avl vector containing an intact E3 region. The reason for this apparent difference in host response between E3- and E3 + Avl vectors is unclear because transactivation o f the E3 region promoter is dependent on E l , which is absent from these types of vector. Studies in non-human primates, including the baboon [39°'], rhesus monkey [38] and macaque (RW Wilmott et al., unpublished data), have demonstrated host responses similar in character to those observed in cotton rats, but with perhaps a more protracted timecourse. Adult baboons receiving 109-1010 pfu per animal o f an
E 1 - / E 3 - Avl vector by bronchoscopic instillation remain clinically well, but show roentgenographic infiltration as well as significant parenchymal cellularity, which peaks 3 weeks after administration [39°°]. Macaques receiving 1010pfu per animal o f an E 1 - / E 3 - A v l vector, delivered bronchoscopically to a single lung lobe, similarly showed no adverse clinical findings, but did present a similar histologic pattern in the lung (R.W Wilmott et al., unpublished data). In rhesus monkeys, intranasal administration o f 2 . 8 x 109pfu per animal of an E1-/E3 + Avl vector resulted in no local or systemic inflammatory response, but did result in anti-vector antibodies [36°°]. Another study in rhesus monkeys reported no histopathology after bronchoscopic delivery of 5 x l 0 9 p f u per animal o f an E 1 - / E 3 - Avl vector to the main bronchi [37]. A third study, in which rhesus monkeys were given 1011 pfu per animal of an Avl vector to the main bronchi, demonstrated histopathology similar to that seen in cotton rats and baboons [38]. A humoral antibody response to Avl vector has been demonstrated in a variety o f animal models, including the cotton rat [12°*], mouse [13°°], baboon [48], macaque [39 °°] and rhesus monkey [36°,37,38]. Pulmonary administration of Avl vectors to cotton rats [12 °°] or macaques (RM Wilmott et al., unpublished data) induces a neutralizing antibody response that is dose-dependent, suggesting an absence of in vivo vector replication. As a control, cotton rats receiving wildtype replicative Ad5 show a similar high-titer antibody response, regardless o f the initial dose [12°°]. Zabner et al. [36°°] have reported that repeated administration o f Avl vectors in cotton rats and rhesus monkeys at rel-
Table 3. Approved Phase I clinical trials of adenovirus-mediated gene therapy for cystic fibrosis lung disease. Vector
Target organ
Dose range (pfu)
Number of patients*
Clinical study site
Principal investigator(s) and references
Ad-CFTR
Nose Lung
2 x 105-2 x 108 2 x 107-2 x 1010
14 10
NIH/University of Cornel l
Crystal [54]
Ad2/CFTR-1
Nose
2 x 106-5 x 107
3
University of Iowa
Welsh [55]
Ad.CB-CFTR
Lung
2.1 x 107-2.1 x1010
20
University of Michigan
Wilson [56]
Avl Cf2
Nose Lung
105-108 106-108
15 15
University of Cincinnati
Wilmott, Whitsett and Trapnell [57,58]
Ad.CB-CFTR
Nose
2 x 108-101!
12
University of North Carolina at Chapel Hill
Boucher and Knowles [59]
Maxillary sinus
2x 107-2x 1010
10-20
University of Iowa
Welsh and Smith**
Ad2-ORF6/PGK-/CFTR-2
*Patient numbers in individual trials are as updated in the Data Management Report from the RAC. **Approved by the National Institutes of Health, February 10th, 1994, Protocol #9312-067
621
622
Mammaliangene studies atively low doses results in successful gene transfer and expression for up to six weeks. Even so, another study has demonstrated that the efficiency of transduction and gene expression after repeat administration is inversely correlated with the humoral antibody response to the vector [12"]. Intravascular administration of Avl vectors results in reporter gene expression in the liver for 60 days in immune deficient athymic (nu/nu) mice compared with only 21 days in normal (CBA) mice, suggesting that a T-lymphocyte response is an important determinant o f the duration o f transgene expression [13**,41°°]. Immunological detection o f low levels of adenoviral hexon and fiber protein supports the concept that basal expression of such adenoviral late gene products in the liver of these animals might provide a basis for a specific cellular immune response directed at the transduced cells [13°°]. This and subsequent studies have verified the existence o f a cytotoxic lymphocyte (CTL) response to adenovirus proteins in animals receiving Avl vectors [13°°]. These observations are supported by studies in normal cotton rats immunocompromized artificially by administration o f dexamethasone or cyclosporin A (S Yei, B Trapnell, unpublished data). O n the basis o f the above observations, a second-generation adenoviral (Av2) vector has now been constructed. A potential problem with Avl vectors is that low-level expression o f adenoviral late genes probably induces an immune attack directly on the transduced cells. As expression of adenoviral late genes is strongly associated with adenoviral D N A replication, the Av2 vector has been designed not only to have deletions in E1 and E3 (similar to Avl), but also to have an additional singlebase substitution in the gene encoding the DNA-binding protein (DBP). This mutation is identical to Ad5ts125, a temperature-sensitive adenovirus mutant with a defect in DBP that renders the virus unable to replicate at 39°C [1,5]. Thus, at a low (permissive) temperature (32°C), the DBP is able to function normally, whereas at the non-permissive temperature (39°C; the core temperature o f rodents), the DBP is non-functional. The inclusion of this mutation in the Av2 vector reduces adenoviral D N A synthesis, late gene expression and cripples replication at the non-permissive temperature. In vivo evaluation o f this Av2 vector delivered to the liver o f normal CBA mice indicates a decreased C T L response [13"',42 °°] and an increased duration o f transgene expression.
Clinical trials with adenoviral vectors At the time of writing, six clinical trials o f human gene therapy using adenoviral vectors have been approved in the United States by the National Institutes o f Health (NIH) Recombinant DNA Activities Committee (RAC) and the Food and Drug Administration (FDA) (Table 3), and one trial has received approval in Europe. All o f these studies involve adenovirus-mediated gene transfer to the respiratory tract of individuals with CE
Final results of one trial involving nasal administration have demonstrated physiological correction o f the chloride secretory defect for three weeks [43•']. No adverse clinical findings linked to the vector itself have been demonstrated. Interim results of another trial have demonstrated the presence o f vector D N A in the nose (seven days after injection) and lung (15 days after injection), expression oftransgene m R N A in the nose at nine days, and C F T R expression in the nose (two days after injection) following vector administration [44°°]. In this study, no clinical, radiological or clinical laboratory findings were observed in patients receiving up to 2 × 107 pfu per patient to the lung. However, one patient receiving 2 × 109 pfu ofadenoviral C F T R in the lung developed an acute self-limited febrile response [44°°]. Although this was associated with a rise in anti-adenovirus antibody titer and lung IL-6 levels, the etiology o f this host-response could not be established definitively.
Conclusions The past year has seen significant and growing interest in the evaluation o f adenoviral vectors in a variety of models o f in uiuo gene therapy. As a result, much is now known concerning the molecular biology of these firstgeneration El-deleted vectors and the host responses that follow in vivo administration, both in a variety o f animal models and in humans. Despite efficient in uivo transgene delivery, expression is transient and appears to be limited by an inflammatory/immune response to the vector. The basis o f this response may include ongoing lowlevel constitutive expression o f adenoviral genes from the vector backbone leading to CTL-induced removal o f transduced cells. A second-generation vector capable o f prolonged transgene expression has been constructed, and it is hoped that this, and other improvements, will further reduce the host response, thus increasing the potential utility of adenoviral vectors.
Acknowledgements We would like to thank jennifer Lee and Soonpin Yei for help with the literature review, Paul Tolstoshev and Elizabeth Ashforth for c~itic}l review of the manuscript, and Gerri Smith for typing the article.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
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2.
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6.
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7. •*
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Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM: Cellular immunity to viral antigens limits El-deleted adenoviruses for gene therapy. Proc Nat/ Acad Sci USA 1994, 91:4407-4411. An important paper describing the T cell mediated host response to in vivo adenovirus administration. This CTL response appears to be directed at transduced cells, leading to their elimination, thus shortening the duration of transgene expression. This study provides a basis for the enhancement of the next generation of adenoviral vectors. 14.
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15. •*
Kay MA, Landen CN, Rotbenberg SR, Taylor LA, Leland F, Wiehle S, Fang B, Bellinger D, Finegold M, Thompson AR et al.: In vivo hepatic gene therapy: complete, albeit transient, correction of factor IX deficiency in hemophilia B dogs. Proc Nat/ Acad Sci USA 1994, 91:2353-2357. Administration of a human factor IX adenovirus corrects the bleeding diathesis in hemophilic dogs. This study demonstrates the feasibility of adenoviral vector mediated gene therapy of a non-rodent animal model for blood dyscrasias resulting from a genetic disorder of liver function. 16. •
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Morsy MA, Alford EL, Bett A, Graham FL, Caskey CT: Efficient adenoviral-mediated ornithine transcarbamylase expression in deficient mouse and human hepatocytes. J C/in Invest 1993, 92:1580-1586. This study describes transient in vivo adenoviral vector mediated correction of the genetic deficiency of ornithine transcarbamylase in the C3H/HENCRMTV mouse, thus demonstrating the feasibility of use of an adenoviral vector for gene therapy for liver disorders. 18.
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19.
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20.
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21.
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22.
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24.
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25.
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26. •
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March KL, Madison JE, Trapnell BC: Pharmacoklnetics of adenoviral vector mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy. Hum Gene Ther 1994, in press. Use of a biocompatible block co-polymer (termed poloxamer 407) can substantially increase the apparent transduction rate constant of an ade-
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31.
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32. *°
Vincent N, Ragot T, Gilgenkrantz H, Couton D, Chafey P, Gregoire A, Briand P, Kaplan JC, Kahn A, Perricaudet M: Long-term correction of mouse dystrophic degeneration by adenovirus-medlated transfer of a mlnldystrophin gene. Nature Genet 1993, 5:130-134. Administration of a minidystrophin gene to the mdx mouse corrects dystrophic muscle degeneration. This demonstrates the feasibility of adenoviral vector mediated gene delivery to correct genetic disorders of muscle.
41. *-
Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson }M: Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nature Genet 1994, 7:362-369. See [42"*].
42. *-
Engelhardt JF, Ye X, Doranz B, Wilson JM: Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci USA 1994, 91:6196-6200. One of several papers (see also [41**]) from the same laboratory describing a single base change within the adenoviral vector backbone that appears to prolong the duration of transgene persistence. This mutation lies within the adenoviral DNA-binding protein gene and confers the temperature-sensitive function to this protein. Presumably, this mutation also blocks the sequential cascade of genetic events leading to low-level adenoviral late gene expression in transduced cells thought to induce a specific cellular immune response. 43. **
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33.
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54.
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36. **
Zabner J, Petersen DM, Puga AP, Graham SM, Couture LA, Keyes LD, Lukason MJ, St George JA, Gregory RJ, Smith AE, Welsh MJ: Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nature Genet 1994, 6:75-83. See [12*°]. 37.
38.
39. °*
Simon RH, Engelhardt JF, Yang Y, Epeda M, Pendleton SW, Grossman M, Wilson JM: Adenovlrus-medlated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum Gene Ther 1993, 4:771-780. An important safety study of human adenovirus vector administration to the lung in non-human primates. This paper helped to alert the scientific community to the importance of the host response to administration of human adenovirus to the lung. 40.
Rosenfeld MA, Chu C, Seth P, Danel C, Banks 1, Yoneyama K, Yoshimura K, Crystal RG: Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replicationdeficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther 1994, 5:331-342.
Gene therapy using adenoviral vectors Trapnell and Gorziglia tion deficient recombinant adenovirus to transfer the normal cystic fibrosis transmembrane conductance regular cDNA to the airway epithelium. Fed Reg 1992, 57:49584. 55.
Welsh MJ (Principal Investigator): Cystic fibrosis gene therapy using an adenovlrus vector: in vlvo safety and efficacy in the nasal epithelium. Fed Reg 1992, 57:49584.
56.
Wilson JM (Principal Investigator): Gene therapy of cystic fibrosis lung diseases using E1 deleted adenovlrus: a phase trial. Fed Reg 1992, 57:49584.
57.
Wilmott RW, whitsett IA, Trapnell BC (Principal Investigators): A Phase 1 study of gene therapy of cystic fibrosis utilizing a replication deficient recombinant adenovlrus vector to deliver the human cystic fibrosis transmembrane conductance cDNA to the airways. Fed Reg 1993, 58:8500-8501.
58.
WilmottRW, Whitsett JA, Trapnell BC: Gene therapy for cystic fibrosis utilizing a replication-deficient recombinant aden-
ovirus vector to deliver the human cystic fibrosis transmembrane conductance regulator cDNA to the airways. Hum Gene Ther 1994, 5:1019-1057. 59.
Boucher RC, Knowles MR (Principal Investigators):Gene therapy for cystic fibrosis using E1 deleted adenovirus: a Phase 1 trial in the nasal cavity. Fed Reg 1993, 58:850].
BC Trapnell and M Gorziglia, Genetic Therapy Inc, 938 Clopper Road, Gaithersburg, Maryland 20878, USA, BC Trapnell, Georgetown University Medical Center, Department of Pharmacology, 3900 keservoir Road, North West Wxshington, District of Columbia 20007-2195, USA. Author for correspondence: BC Trapnell at Genetic Therapy Inc.
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